International Journal of Agriculture and Forestry 2012, 2(4): 150-156 DOI: 10.5923/j.ijaf.20120204.03

Phylogenetic Relationships of Santalum album and its Adulterants as Inferred from Nuclear DNA Sequences

Anupama Chembath1, Balasundaran M1,*, Sujanapal P2

1Forest Genetics and Biotechnology Division, Kerala Forest Research Institute, Peechi, Thrissur, Kerala, 680653, India 2Sustainable Forest Management Division, Kerala Forest Research Institute, Peechi, Thrissur, Kerala, 680653, India

Abstract The East Indian sandalwood, Santalum album, valued for its fragrant oil yielding heartwood is a major ingre- dient in indigenous medicines and perfumes. Scarcity of sandal has led to illegal felling of sandal trees, and adulteration of sandalwood and oil. This study represents the first molecular phylogeny of S. album and its adulterant species wightiana, Erythroxylum monogynum, Buxus sempervirens, Ximenia americana, , and Chukrasia tabularis through 18S and 26S rDNA sequencing. In the Maximum Parsimony (MP) tree for 18S and 26S rDNA data sets, moderate to high bootstrap support was obtained for the nodes. For 18S rDNA data sets, the tree had B. sempervirens and X. Americana as the upper branch, with E. monogynum branched separately to the cluster. The lower branch had S. album and O. wightiana with O. lanceolata joining separately to both clades of the tree. In the MP tree for 26S rDNA datasets, S. album and O. wightiana formed the major cluster with X. americana clustering separate and B. sempervirens and O. wightiana as the lower branch with C. tabularis clustering separate to the tree. The molecular data presented here provided useful information for resolving the phylogenetic relationship of these . Inferences from this study are in accordance with Cronquist’s system of classification of flowering plants where all the species originate from a single phylogenetic tree of Rosidae. Keywords Santalum Album, Adulterants, Gene Sequencing, Sequence Comparison, Molecular Phylogeny

For this reason, sandalwood is often adulterated using 1. Introduction Osyris spp. Osyris lanceolata Hochst. & Steud., a member of family, also called Tanzanian Sandalwood or Santalum album L., commercially known as East Indian East African Sandalwood, possesses scented heart wood. sandalwood is a medium sized, xylem tapping, root Trees of the genus Osyris are either shrubs or small ever- hemi-parasitic tree belonging to the family Santalaceae. The green trees, and are usually root hemi-parasites. But the oil, species, commonly known as sandal is valued for its heart- used in pharmaceutical and cosmetic industries abroad, lacks wood containing the precious sandal oil. The Indian san- the sensuality of East Indian sandalwood oil. dalwood has the highest oil content (6 to 7%) and a desirable O. wightiana Wall. ex Wight. is found to occur rarely in aroma profile, highly prized in perfumery and indigenous higher altitudes (900 m above m.s.l.) of Idukki district of medicine[1]. Kerala state[4] and in Tamil Nadu[5] in India. Erythroxylum The annual production of sandalwood worldwide is esti- monogynum Roxb., possessing fragrant heartwood, native to mated between 200 to 300 tonnes, of which 90 per cent is the Indian subcontinent is the source of ‘Indian bastard from India. Scarcity of sandal in open market and consequent sandal’, also used to adulterate sandalwood[6]. Erythroxy- price hike to exorbitant level have led to illegal felling and laceae comprises 200 species distributed throughout the smuggling of trees. Sandal has been categorized as ‘Vul- tropics. The root bark contains alkaloids; the heartwood is nerable’ in the Red Data List by IUCN[2]. Due to its com- light brown which is very durable and easy to work. mercial importance, sandalwood arriving for trade in the Other scented woods often used to adulterate sandalwood market is adulterated with many other indigenous as well as are Buxus sempervirens L., Ximenia americana L. and imported scented wood species. Chukrasia tabularis var. velutina. B. sempervirens, com- Rao et al.[3] have listed a few timbers that are used for monly known as American boxwood, a member of the adulterating sandalwood. Wood of S. album and Osyris Buxaceae family is an evergreen shrub native to Western and species are strikingly similar in most of the wood anatomical Southern Europe, Northwest Africa and Southwest Asia[7]. characters. Wood is very hard and heavy, used for engraving, marquetry and wood turning. X. Americana, belonging to Olacaceae * Corresponding author: [email protected] (Balasundaran M) family, also known as ‘false sandalwood’ is a small, shrubby Published online at http://journal.sapub.org/ijaf tree native to Central and South Florida and the African Copyright © 2012 Scientific & Academic Publishing. All Rights Reserved tropics[8]. It has hemi-parasitic roots, but it does not require a

International Journal of Agriculture and Forestry 2012, 2(4): 150-156 151

host to thrive. Bark and roots are used for tanning and wood were synthesized at MWG Biotech Pvt. Ltd., Bangalore, for firewood and charcoal. C. tabularis, a member of the India. Using these primers, we PCR amplified 18S and 26S Meliaceae family, locally known as ‘agil’ in Kerala, is a rDNA of S. album and O. wightiana using DNA from leaf deciduous medium-sized tree found in India, Bangladesh, samples of the species collected from Marayur as template. China, Thailand and Malaysia[9]. Freshly cut wood has a The primer sets used for amplifying O. wightiana loci were fragrant odour, but dried wood has no characteristic odour. used for the amplification of loci from E. monogynum, col- Planed surfaces have a high lustrous satiny sheen. The timber lected from the same area. is highly prized for superior cabinet work, decorative panel- ling, interior joinery, carving, toys and turnery. 2.3. PCR Amplification and Sequencing The state of knowledge about relationships among the PCR amplification reactions were performed using various lineages of land plants is currently incomplete[10]. FINNZYMES High Fidelity PCR Kit. The PCR products Clarification of phylogenetic relationships among were subjected to sequencing at MWG Biotech Pvt. Ltd. The and their adulterant groups presents opportunities to better 26S rDNA region of E. monogynum could not be amplified; understand their evolutionary and interfamilial relationships. hence it was not included in the present study. The se- Ribosomal RNA or DNA sequences (rRNA/ rDNA) have quencing was performed using BigDye terminator v3.1 cycle frequently been used to reconstruct deep branches of evolu- sequencing Kit containing AmpliTac DNA polymerase tionary history. The gene containing highly conserved as (Applied Biosystems). well as variable regions facilitate alignments of nucleotide sequences derived from phylogenetically linked taxa[11]. Table 1. 18S and 26S rDNA primers designed and developed for S. album, E. monogynum and Osyris sp. 18S rRNA/rDNA has been used for phylogeny reconstruc- NCBI Tm( ) tion within many groups of eukaryotes[12]. Although phy- Ac- and logenetic analysis of 18S rDNA sequences provides criti- Primer Sequence (5'-3') ces- Product℃ sion cally independent data set for the assessment of higher-level size(bp) relationships, in many instances analysis of 18S rDNA se- No 57.3 18SA/F TCCTGCCAGTAGTCATATGC FJ58 quence alone will not provide adequate resolution. Com- 1695 8874 bining 26S rDNA sequence data and 18S rDNA sequence 18SA/R AAGGTTCAGTGGACTTCTCG 57.3 55.9 data would increase the quantum of phylogenetically in- 18OL/F GTCTCAAAGATTAAGCCATGC FJ58 1668 8875 formative history fourfold and provide greater resolution and 18OL/R TAAGGTTCAGTGAACTTCTCG 55.9 support at higher taxonomic levels [13]. The present inves- 55.9 18ER/F GTCTCAAAGATTAAGCCATGC FJ58 1587 tigation has been designed to deduce the evolutionary rela- 8876 tionships among S. album and its adulterant wood species 18ER/R TAAGGTTCAGTGAACTTCTCG 55.9 54.0 26SA/F CCGTTGAGTTTAAGCATATCA FJ58 using rDNA sequencing and comparison studies. 3204 8877 26SA/R TACCCTGTCGCATATTTAAGT 54.0 57.3 26OL/F GTTACCCGCTGAGTTTAAGC FJ58 3264 8878 2. Materials and Methods 26OL/R CACTCTGCCGCTTACAATAC 57.3

2.1. Sample Collection and DNA Extraction 2.4. Phylogenetic Analyses

Semi-dry logs and leaf samples of Santalum album were Phylogenetic analyses were conducted using Molecular collected from Marayur Sandalwood reserve forest located Evolutionary Genetics Analysis (MEGA) software version on the leeward side of the Western Ghats in the South West 4[15]. Sequences were manually aligned using Alignment region of India. Samples of Osyris wightiana and Explorer (AE) in MEGA 4 and separate alignments were Erythroxylum monogynum were collected from Chinnar created for 18S and 26S rDNA data sets. Sequences of 18S wildlife sanctuary located adjacent to the Marayur sandal and 26S rDNA of sandalwood adulterants Buxus semper- reserve forest. All these places lie at 10º15’ N latitude and virens (L54065, AF389243), Ximenia americana (L24428, 77º11’ E longitude. DNA extractions were accomplished DQ790220) and Osyris lanceolata (U42803, AF389274), using QIAGEN DNeasy Mini Kit and the DNA was and 26S rDNA of Chukrasia tabularis (AY128154) depos- used as template for PCR amplification of ribosomal DNA ited in the NCBI nucleotide library were chosen as outgroups using specific primers designed and developed for Santalum and were included in the alignment. The 18S rDNA sequence and Osyris species. of C. tabularis was unavailable in the NCBI nucleotide li- brary; hence it was not included for the analysis. 2.2. Primer Design and Synthesis The evolutionary history was inferred using the Maximum From the 18S and 26S ribosomal RNA partial sequences Parsimony (MP) method[16]. The consistency index, reten- of S. album (L24416, AY957453) and Osyris lanceolata tion index and the composite index for all sites and parsi- (U42803, AF389274) deposited in the NCBI nucleotide mony-informative sites were calculated. The MP tree was library, specific primers capable of amplifying partial ribo- obtained using the Close-Neighbour-Interchange algo- somal DNA units were designed [14] (Table 1). The primers rithm[17] with search level 2 in which the initial trees were

152 Anupama Chembath et al.: Phylogenetic Relationships of Santalum album and its Adulterants as Inferred from Nuclear DNA Sequences obtained with the random addition of sequences (100 repli- positions, out of which 1198 were parsimony informative. cates). All positions containing gaps and missing data were The consistency index was 0.7480, the retention index eliminated from the dataset using complete deletion option. 0.3735, and the composite index 0.2975 for all sites and Nucleotide pair frequencies including identical, transitional parsimony-informative sites. Likewise, for 26S rDNA and transversional pairs and the overall transi- dataset there were a total of 740 positions in the final dataset, tion/transversion bias were estimated and substitution pat- out of which 540 were parsimony informative. The consis- terns were calculated. Tajima’s relative rate test for testing tency index was 0.7827, the retention index 0.5135, and the molecular clock hypothesis was performed to test the con- composite index was 0.4278 for all sites and parsimony stancy of evolutionary rates between two sequences or informative sites. clusters of sequences, using an outgroup sequence. P-value Nucleotide pair frequency estimations in 18S and 26S and χ2 test statistics were calculated with one degree of rDNA sequences, results of substitution pattern calculations freedom[18]. with S. album as the reference sequence, and the overall In the bootstrap test of phylogeny using Maximum Par- transition/transversion bias are provided in Table 2. simony method, the consensus tree inferred from 1000 rep- Tajima’s relative rate test for testing molecular clock licates were taken to represent the evolutionary history of the hypothesis in 18S and 26S rDNA sequences was performed taxa analyzed[19]. The percentage of replicate trees in which with S. album (lineage 1) kept constant (Table 3). the associated taxa clustered together in the bootstrap test In the bootstrap test of phylogeny using Maximum Par- (1000 replicates) was shown next to the branches of the out simony (MP) method the bootstrap consensus tree inferred tree. from 1000 replicates was taken to represent the evolutionary history of the taxa analysed. The percentage of replicate trees 2.5. Sequence Comparison and Similarity Search in which the associated taxa clustered together in the boot- Comparison of 18S and 26S rDNA sequences of S. album, strap test is shown next to the branches of the out tree. In the O. wightiana and E. monogynum with outgroup sequences of MP tree for 18S rDNA data sets (Figure 1) the most parsi- B. sempervirens, X. americana, O. lanceolata and C. tabu- monious tree was with length 4272. Moderate to high boot- laris were achieved through the bioinformatics tool for strap support was obtained for the nodes, and the lower multiple sequence alignment, CLUSTAL W (1.83). Se- branch formed a clade (A) of Santalales with S. album and O. quence similarity search for 18S and 26S rDNA sequences of wightiana (75% BS) clustered together. The upper branch S. album, O. wightiana and E. monogynum were accom- had high BS support (100%) for B. sempervirens and X. plished by means of NCBI-BLAST. Americana (Clade B), and also had E. monogynum sepa- rately branched to that cluster, but with low BS support (<50%). O. lanceolata joined separately to both the clades of 3. Results the tree. Aligned datasets for 18S rDNA genes contained 1554

Table 2. Nucleotide pair frequencies and substitution pattern calculations in 18S and 26S rDNA sequences with S. album as reference Sequence name Identical pairs Transitional pairs Transversional pairs Overall Transition/ Transversion bias (R) 18S rDNA O. wightiana 428 435 805 3.910 E. monogynum 401 386 800 3.606 B. sempervirens 443 416 806 3.398 X. americana 427 441 822 3.622 O. lanceolata 439 436 820 3.385 26S rDNA O. wightiana 856 784 1564 3.966 B. sempervirens 775 790 1551 4.270 O. lanceolata 808 770 1626 3.796 X. americana 220 223 391 5.061 C. tabularis 235 193 447 3.927

Table 3. Tajima’s relative rate test for testing molecular clock hypothesis in 18S and 26S rDNA sequences with Lineage 1 (S. album) kept constant Lineage 1 Lineage 2 Outgroup Divergent sites P-value χ2 test statistic 18S rDNA S. album O. wightiana E. monogynum 614 0.2367 1.40 S. album E. monogynum B. sempervirens 531 0.7815 0.08 S. album B. sempervirens X. americana 421 0.0000 169.7 S .album X. americana O. lanceolata 632 0.6614 0.19 S. album O. lanceolata O. wightiana 616 0.7791 0.08 26S rDNA S. album O. wightiana B. sempervirens 1165 0.3536 0.86 S. album B. sempervirens O. lanceolata 963 0.0000 113.8 S. album O. lanceolata X. americana 292 0.8287 0.05 S. album X. americana C. tabularis 301 0.4581 0.55 S. album C. tabularis O. wightiana 301 0.0922 2.83

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Buxus sempervirens 100 36 Clade B 36 Ximenia americana

Erythroxylum monogynum

Clade A Santalum album 7 75 Osyris wightiana

Osyris lanceolata

Figure 1. Bootstrap consensus tree (MP) of 18S rDNA dataset. Numbers above nodes are bootstrap percentages (1000 replicates)

Santalum album 77 Clade C Osyris wightiana 57

Ximenia americana

Buxus sempervirens Clade D 100 Osyris lanceolata

Chukrasia tabularis

Figure 2. Bootstrap consensus tree (MP) of 26S rDNA dataset. Numbers above nodes are bootstrap percentages (1000 replicates)

Table 4. 18S and 26S rDNA Sequence comparison by CLUSTAL W (1.83) for multiple sequence alignment scores Identity Score Sl No Sequence A Sequence B 18S rDNA 26S rDNA 1 S. album O. wightiana 98 92 2 S. album E. monogynum 96 - 3 S. album B. sempervirens 94 87 4 S. album O. lanceolata 98 92 5 S. album X. americana 97 74 6 S. album C. tabularis - 62 7 O. wightiana E. monogynum 96 - 8 O. wightiana B. sempervirens 94 90 9 O. wightiana X. americana 97 86 10 O. wightiana O. lanceolata 99 98 11 O. wightiana C. tabularis - 92 12 B. sempervirens X. americana 95 77 13 B. sempervirens O. lanceolata 95 91 14 B. sempervirens C. tabularis - 82 15 X. americana O. lanceolata 97 88 16 X. americana C. tabularis - 61 17 O. lanceolata C. tabularis - 92 18 E. monogynum B. sempervirens 97 - 19 E. monogynum X. americana 96 - 20 E. monogynum O. lanceolata 96 -

154 Anupama Chembath et al.: Phylogenetic Relationships of Santalum album and its Adulterants as Inferred from Nuclear DNA Sequences

In the MP tree for 26S rDNA datasets (Figure 2) the most was resolved comprising S. album and O. wightiana (BS parsimonious tree was with length 1840. MP resolved a 77%) with X. americana clustering separately (BS 57%). monophyletic Santalales (Clade C) consisting of S. album Clade D was found to be polyphyletic and had high BS and O. wightiana (BS 77%) in the major cluster with X. support (100%) for B. sempervirens and O. lanceolata. C. americana clustering separate (BS 57%). The next clade (D) tabularis clustered separate and was connected to both the had high BS support (100%) for B. sempervirens and O. clades of the 26S MP tree. In the phylogenetic system of wightiana. C. tabularis clustered separate to the 26S MP classification,[22] all the species belong to the division tree. Magnoliophyta and class Magnoliopsida. Except B. sem- Comparisons between 18S and 26S rDNA sequences and pervirens all other species belong to the subclass Rosidae of their outgroups using bioinformatics tool CLUSTAL W phylogenetic tree. B. sempervirens belongs to the subclass (1.83) for multiple sequence alignment gave scores indica- Hamamelididae which is primitive than the subclass Dil- tive of the identities and differences between the sequences lenidae and Rosidae in the phylogenetic hierarchy and is the (Table 4). most primitive among all other species selected for the study. In the 18S sequence scores the most identical species to- Among the Rosidae members, S. album, O. wightiana, O. wards S. album were O. wightiana and O. lanceolata giving lanceolata and X. americana belong to the order Santalales; a score of 98, and the least identical being B. sempervirens within them the closest being Santalaceae and Olacaceae. with a score of 94. At the same time, for 26S rDNA sequence Olacaceae is considered as the most primitive among San- scores also, the most identical scores were for O. wightiana talalean families as it comprises both root parasitic and and O. lanceolata with 92, while the least one being C. nonparasitic species. Kuijt[23] considered the Olacaceae the tabularis with 62 as the identity score. Plexus from which the other Santalalean families were de- rived. E. monogynum the immediate relative of Santalanae group belonged to the Linales under Geraninae and is the 4. Discussion most primitive of all the above species. While considering all This study represents the first molecular phylogeny of S. the species selected for the study C. tabularis of the order album and its adulterant species inferred from 18S and 26S Rutales of Rutanae group is the primitive among the Rosidae rDNA sequences. The molecular data presented here, though members. limited in number, provides novel information useful in Study of phylogenetic relationships of Santalales and their resolving the phylogenetic history of these plants. By fol- relatives by Nickrent and Franchina using 18S and 26S lowing Bentham and Hooker[20] with recent circumscription rDNA sequences indicated that sequence of Buxus when of certain families to their current concept[21], all the species compared with several genera of Rosidae, the genus nested selected for the study belong to the class Dicotyledonae of within the clade composed of Cornales and Apiales and not natural system. In all the cases, the floral lobes are 3-6 with in a basal position near or outside Fabales. Analyses by perianth, with or without calyx, and corolla lobes which are Nickrent et al. have placed Ximenia in the clade D of Ola- free or connate. Principal species, S. album and its closest caceae suggesting that haustorial parasitism arose just once natural relatives O. wightiana and O. lanceolata belong to in Santalales. In their study, Santalum and Osyris clustered the family Santalaceae under the order Santalales belonging together forming a well supported clade (clade E) of San- to the subclass Monochlamydeae. B. sempervirens belonging talaceae. to the family Buxaceae is the next advanced relative in the The molecular evidences presented in a study by Der and natural hierarchy. In the natural system of classification, Nickrent[24] corroborated the polyphyletic nature of San- Santalaceae and Balanophoraceae of the order Unisexuales taleae and illustrated Santalum being strongly supported in Monochlamydeae are immediately followed by Buxaceae clade, but relationships among its various subclades some- and Euphorbiaceae. times being poorly resolved. Phylogenetic relationships In the 18S MP tree, monophyletic Clade A of Santalales among early-diverging based on four genes grouped consisted of S. album, O. wightiana (75% BS) clustered Osyris into core eudicots and Buxus into early diverging together. A polyphyletic Clade B composed of B. semper- eudicots[25]. virens and X. americana (100% BS) and E. monogynum A revised classification of Santalales was presented by separately branched to that cluster (BS <50%). O. lanceolata Nickrent et al[26] in which the molecular and morphological branched separately and joined to both the clades of the 18S data on santalalean clades were drawn together and a revised MP tree. Among the species selected for the study S. album, classification for the entire order was proposed. They came O. wightiana, O. lanceolata and B. sempervirens are con- across several instances in which support values along the sidered as advanced amongst Dicotyledonae. Species such as portions of the molecular tree were low (polytomies), thus E. monogynum and X. americana are in the primary primi- not providing full resolution of interfamilial relationships. tive group in hierarchy of class Dicotyledonae. Hence, these They also suggest that the tendency for monogeneric groups species are broadly categorized into two clusters- one ad- to occur as sister to species-rich clades is not unusual within vanced and the other primitive. angiosperms, and in such situations, circumscribing mono- In the 26S MP tree, a monophyletic Santalales (Clade C) generic families appears to be the best solution.

International Journal of Agriculture and Forestry 2012, 2(4): 150-156 155

Angiosperm phylogeny by Soltis et al[27] revealed that similarity search using NCBI-BLAST. Phylogenetic analy- relationships among major clades of Santalales have not ses were done using Molecular Evolutionary Genetics been resolved with strong BS support in recent analyses, Analysis (MEGA) software. In the Maximum Parsimony and many segregate families have been newly recognized. (MP) tree for 18S and 26S rDNA data sets, moderate to high An angiosperm phylogeny was reconstructed using four bootstrap support was obtained for the nodes. While con- slowly evolving mitochondrial genes by Qiu et al[28] in sidering all the species selected for the study, C. tabularis of which S. album and X. americana clustered together within the order Rutales is the primitive among the Rosidae mem- Santalales, which is a member of an expanded asterid bers. Inferences from the present study are in accordance clade. B. sempervirens clustered within Buxales which with the Cronquist’s classification of flowering plants (1988) forms a basal grade that diverged before the diversifica- where all the species originate from a single phylogenetic tion of asterids. tree of Rosidae and Chukrasia tabularis is the distant relative. Phylogenetics of early branching eudicots were examined Cladistic data provided more insight into the phylogenetic by Barniske et al[29] which also placed Buxales as succes- relationships of these biochemically related taxa in several sive sisters to core eudicots. In a study by Moore et al[30] aspects. which looked at the phylogenetic analysis of plastid genes, Maximum-likelihood analyses of the gene alignment placed Santalales as successive sisters to Asteridae, with each node ACKNOWLEDGEMENTS receiving 99-100% Bootstrap support. We are grateful to the Kerala Forest Department for per- However, in Cronquist system[31], all the species origi- mission to collect the samples. The study was funded by nate from a single phylogenetic tree of Rosidae and Kerala State Council for Science, Technology and Envi- Chukrasia tabularis is the distant relative. Earlier workers ronment. recognized the polyphyletic relationship of subclass Rosidae and its orders such as Santalales. These selected diverse group of species showed different mode of nutrition and habitat not only owing to its phylogeny, but also due to ad- aptation or parallel evolution. Results of the present study are REFERENCES in agreement with the Cronquist classification and the [1] Sanjaya, Ananthapadmanabha H S, Ravishankar Rai, V, “In cladistic data provided more insight into the phylogenetic vitro shoot multiplication from the mature tree of Santalum relationships of these biochemically related taxa in several album L.” in Sandal and Its Products, ACIAR Proceedings No. aspects. Though, a wider sampling and information on se- 84. 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156 Anupama Chembath et al.: Phylogenetic Relationships of Santalum album and its Adulterants as Inferred from Nuclear DNA Sequences

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